This revised proposal addresses the provocative question of how mitochondrial heterogeneity influences tumorigenesis or progression (NCI PQ5). We will use single-cell, quantitative measurements to characterize the status of the mitochondrial network (proteins involved in mitochondrial dynamics and apoptosis) in glioblastoma multiforme (GBM). We hypothesize that the subpopulations of glioma stem-like cells (GSCs) with increased mitochondrial division will display increased self-renewal capacity and increased apoptotic resistance to routinely used chemotherapy. Our prediction is that the combination of all these properties makes this population of cells highly tumorigenic. GSCs are hypothesized to underlie tumor recurrence - which is inevitable in 95% of these patients, making it a significant problem in GBM. These single-cell studies could reveal new properties of the glioblastoma-propagating cells that have been overlooked by population-based approaches. The proposed approach will integrate information at the single-cell level into the overall complexity of the tumor environment. Our goal is to understand the molecular basis of the heterogeneity of mitochondrial dynamics/function that may underlie differences in the response to therapy. In Aim 1, we will use single-cell measurements of mitochondrial activity and function (mitochondrial dynamics and apoptotic-related proteins) together with stem-like markers to acquire a global profile of the mitochondrial heterogeneity in GSCs, using mass cytometry. Following this phenotypic characterization, we will examine the positional identity of the newly identified cell subpopulations within the tumor microenvironment using brain tumor organoids. This approach will be amenable to longitudinal assays for following tumor progression in the dish. In Aim 2, we plan to reveal the heterogeneity of the mitochondrial status in GBM by two strategies. First, we will assess the priming state of the GBM tumors by examining their sensitivity to conventional chemotherapy, using dynamic BH3 profiling. This will provide an inter-tumoral assessment of heterogeneity. Second, we will isolate specific subpopulations of cells (e.g. stem-like/high or low mitochondrial fission/fusion) and assess their sensitivity to conventional chemotherapy and/or inhibitors of the mitochondrial dynamics machinery. Completion of these Aims will provide valuable information at the single-cell level about the contribution of mitochondrial heterogeneity to tumorigenesis and drug resistance in GBM. This information will provide the foundation for future studies aimed to improve current chemotherapy regimens and to rationalize potential new therapies.